With the extension of the human life span, women spend one-third of their lives beyond the reproductive years. The transition into menopause, a normal process of aging, is associated with physical risks and psychological adjustments. It is critical to improve understanding of the these changes. However, there is a lack of diagnostic tools for monitoring the temporal stages in the history of menopause despite the importance of this transition period. There is no reliable test to determine how close a woman is to menopause. Clinical decisions for treatment of perimenopausal women today are based chiefly upon subjective symptoms rather than objective diagnostic tests.
There is a lack of adequate chemical markers for defining the menopausal state since neither serum gonadotropins, estradiol, nor inhibin A or B levels are adequate for diagnosis unless daily sampling is performed for prolonged periods of time (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Hee, et al. 1993; Metcalf, 1988). A number of studies of women during the periovulatory period have indicated that the currently used biochemical markers of menopause are inadequate (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Hee, et al. 1993; Metcalf, 1988; Santoro, et al. 1996). Gonadotropin levels fluctuate from postmenopausal concentrations back down to levels found in normal, young cycling women (Burger, 1996; Burger, et al., 1995; Burger, 1994a; Metcalf, 1988; Metcalf, et al. 1982; Metcalf and Donald, 1979). What appear to be normal ovulatory cycles may follow prolonged anovulatory periods coincident with postmenopausal concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Metcalf, 1988; Metcalf, and Donald, 1979; Metcalf, et al. 1981a). Some investigators declare that all current biochemical measurements have little predictive value during the menopausal transition because of the great variations in levels of steroids and gonadotropins. (Burger, 1996;. Burger, 1994a; Burger, 1994b; Hee, et al. 1993;. Metcalf, and Donald, 1979; Metcalf, et al. 1981b; Metcalf, 1979).
Although elevations in certain serum gonadotropin levels reflecting gametogenic failure usually occur several years before a decline in estrogen and irregular cycling begins, measurement of serum gonadotropin levels, estrogen, and inhibins A and B have limited value to the practicing physician. A reliable test is essential to differentiate a premenopausal woman from a woman very early in perimenopause or the latter from one in the middle of the transition; menopausal changes could be placed in relation to the stage of menopausal transition. This would help to resolve, for example, whether treatment for osteoporosis should begin much earlier or that hormone replacement therapy should begin at a different time rather than based on symptomatic discomfit. The present invention solves these problems by providing urinary-based immunoassay methods and assay kits.
Human gonadotropins undergo metabolic transformations, which result in the presence of several smaller, structurally and immunologically related forms in the urine. A major form of urinary hCG-associated immunoreactivity is an epitope on a molecule smaller than heterodimeric hCG (Birken et al., 1996; O'Connor et al., 1994; Schroeder and Halter, 1983). This molecule has been identified as an hCG beta core fragment (hCGβcf) (Birken et al., 1988; Blithe et al., 1988). In normal pregnancy, the core fragment constitutes a major mole fraction of urinary hCG excretion (Kato and Braunstein, 1988). Using polyclonal antisera raised against hCGβcf, immunoreactive beta core like activity can be detected in both postmenopausal women and in the periovulatory period of the normal menstrual cycle (Iles et al., 1992; Neven et al., 1993). However, some immunoreactivity results from cross-reactivity with the polyclonal hCGβcf antibodies. An hLH beta core fragment (hLHβcf) has been isolated from human pituitaries and a panel of monclonal antibodies has been generated (Birken et al., 1993a; Kovalevskaya et al., 1995).
The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physico-chemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of hLHβcf have a different structure, probably in the carbohydrate moieties. The carbohydrate moiety of the pituitary hLHβcf resembles that of pituitary hLHβ rather than displaying the degraded carbohydrate chains present in urinary hCGβcf. The endogenous urinary core material is extremely stable to repeated freeze/thaw cycles and prolonged storage at 4° C. or at room temperature. HLHβcf cross-reaction with some hLH or hLHβ monoclonal antibodies may well interfere with the accurate estimation of the day of hLH surge when urinary tests are utilized. The expression of hLHβcf in the urine of both reproductive and postreproductive age women and in men, is now characterized employing assays highly specific for the pituitary form of the fragment.
Analysis of the metabolites of the gonadotropins excreted into urine can help to distinguish between healthy and abnormal physiological states. For example, the hCG β core fragment (hCGβcf) is present at high levels in the urine of normal pregnant women (Kato et al., 1988) but, also, occurs abnormally in the urine of nonpregnant patients with a variety of malignancies (O'Connor et al., 1988, Cole et al., 1988a, 1988b, 1990). Until now, it has not been possible to distinctly measure one of the fragments in the presence of the others. For example, the utility of the hCGβcf molecule as a marker of malignancies in postmenopausal women has been compromised by the cross-reactions of antibodies elicited to the hCGβcf with a molecule of similar structure and size (presumably the homologous fragment of hLH) excreted by normal postmenopausal women in their urine. Consequently, the high threshold measurement compromised the ability of hCGβcf to serve as a cancer marker in this important patient population. Distinguishing hLHβcf from an hCGβcf, therefore, is of great value. A preponderance of hLHβcf may indicate a normal state while a major mole fraction of the hCG fragment may be associated with malignancy (Birken et al., 1993b). The present invention provides a method to make such a distinction. Immunological analysis of the hLHβcf in normal cycling women, as compared with infertile patients, may identify a metabolic marker associated with an abnormal state (i.e.an ovulatory cycles, polycystic ovarian disease). Antibodies to the hLHβcf, isolated from pituitary extract, can also be used to measure such a molecule in urine.
Methods for specific immunometric assays which report the levels of expression of this new hLH molecular form, hLHβcf, in men and women at different stages of their reproductive history are described herein. The present invention now makes it possible to evaluate the metabolism of hLH in premenopausal, perimenopausal and postmenopausal women and in men and to distinguish between normal and abnormal physiological states.
In addition, these methods to visualize LH fragment in plasma differentiates LH fragment derived directly from pituitary from that derived by peripheral cleavage of LH. hLHβcf may circulate in plasma.
The methods described herein measure the stable metabolic products of LH which are excreted into urine usually at much higher concentrations than the parent hormones, themselves, are found in urine or blood. These assays do not use heterodimeric hormones which are unstable, unless supplemented by stabilizers such as glycerol, because they dissociate into their constituent, non-covalently bound subunits, especially under acid conditions or upon freeze thaw cycles. Urinary metabolic forms represent end-products of a degradative process. The forms explored have proven to be stable unlike the parent hormones which can dissociate into free subunits greatly complicating urinary measurements. Antibodies specific for hLH beta core fragment some of which are referred to in the present application, have been detailed in the related co-pending U.S. application Ser. No. 08/763,669 filed Dec. 11, 1996, the content of which is hereby incorporated by reference.
In particular, related co-pending U.S. application Ser. No. 08/763,669 filed Dec. 11, 1996, describes monoclonal antibody designated B505. The hybridoma cell lines for producing the antibody designated B505 and an antibody designated B503 were deposited pursuant to and in satisfaction of the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas Va. 20110-2209, on Dec. 11, 1995 under ATCC Designation Nos. 12000 and 11999, respectively. All restrictions upon public access to these documents shall be removed upon the grant of a patent on this application and the deposit shall be replaced if viable samples cannot be made by the depository named hereinabove.
The present invention takes advantage of the natural metabolic processing of LH as a means of improving the diagnosis of women in perimenopause as well as to assess patterns of metabolites useful for monitoring estrogen replacement therapy.
An antibody, designated B152, produced by the hybridoma cell accorded ATCC Accession number HB-12467, generated against a nicked form of hCG isolated from a choriocarcinoma patient, but not specific for nicked isoform hCG is able to discriminate among carbohydrate variants of hCG. B152 is specific for an early pregnancy associated molecular isoform of hCG.which in the first four weeks of pregnancy is measured at much higher quantities than the hCG standard isoforms measured by the usual heterodimeric hCG assays exemplified by a previously decribed B109 based assay. Later in pregnancy, the B152 isoform declines and is lower in third trimester pregnancy urine than the standard isoforms measured by B109.
Because of its presence in urine, measurement of hLHβcf greatly adds to the sensitivity of measurements for the presence of related molecules in early pregnancy as well as in malignancies, including malignancies in postmenopausal women and non-trophoblastic cancers. Its measurement may be useful for prediction of Down's syndrome pregnancies and ectopic pregnancies. The core fragment of hLHβ is useful as a urinary marker for many different physiological states including disease, as markers of the state of senescence the ovary.